Engineering of Serine-Deamination pathway, Entner-Doudoroff pathway and pyruvate dehydrogenase complex to improve poly(3-hydroxybutyrate) production in Escherichia coli.

BACKGROUND: Poly(3-hydroxybutyrate) (PHB), a biodegradable bio-plastic, is one of the most common homopolymer of polyhydroxyalkanoates (PHAs). PHB is synthesized by a variety of microorganisms as intracellular carbon and energy storage compounds in response to environmental stresses. Bio-based production of PHB from renewable feedstock is a promising and sustainable alternative to the petroleum-based chemical synthesis of plastics. In this study, a novel strategy was applied to improve the PHB biosynthesis from different carbon sources. RESULTS: In this research, we have constructed E. coli strains to produce PHB by engineering the Serine-Deamination (SD) pathway, the Entner-Doudoroff (ED) pathway, and the pyruvate dehydrogenase (PDH) complex. Firstly, co-overexpression of sdaA (encodes L-serine deaminase), L-serine biosynthesis genes and pgk (encodes phosphoglycerate kinase) activated the SD Pathway, and the resulting strain SD02 (pBHR68), harboring the PHB biosynthesis genes from Ralstonia eutropha, produced 4.86 g/L PHB using glucose as the sole carbon source, representing a 2.34-fold increase compared to the reference strain. In addition, activating the ED pathway together with overexpressing the PDH complex further increased the PHB production to 5.54 g/L with content of 81.1% CDW. The intracellular acetyl-CoA concentration and the [NADPH]/[NADP(+)] ratio were enhanced after the modification of SD pathway, ED pathway and the PDH complex. Meanwhile, these engineering strains also had a significant increase in PHB concentration and content when xylose or glycerol was used as carbon source. CONCLUSIONS: Significant levels of PHB biosynthesis from different kinds of carbon sources can be achieved by engineering the Serine-Deamination pathway, Entner-Doudoroff pathway and pyruvate dehydrogenase complex in E. coli JM109 harboring the PHB biosynthesis genes from Ralstonia eutropha. This work demonstrates a novel strategy for improving PHB production in E. coli. The strategy reported here should be useful for the bio-based production of PHB from renewable resources.

Localization of polysome-bound albumin and serine dehydratase in rat liver cell fractions.

The polysomes involved in albumin and serine dehydratase synthesis were identified and localized by the binding to rat liver polysomes of anti-rat serum albumin and anti-serine dehydratase [(125)I]Fab dimer and monomer. Techniques were developed for the isolation of undegraded free and membrane-bound polysomes and for the preparation of [(125)I]Fab monomers and dimers from the IgG obtained from the antisera to the two proteins, rat serum albumin and serine dehydratase. The distribution of anti-rat serum albumin [(125)I]Fab dimer in the polysome profile is in accordance with the size of polysomes that are expected to be synthesizing albumin. By direct precipitation, it has been demonstrated that nascent chains isolated from the membrane-bound polysomes by puromycin were precipitated by anti-rat serum albumin-IgG at a level of 5-6 times those released from free polysomes. Anti-rat serum albumin-[(125)I]Fab dimer reacted with membrane-bound polysomes almost exclusively compared to the binding of nonimmune, control [(125)I]Fab dimer; a significant degree of binding of anti-rat serum albumin-[(125)I]Fab to free polysomes was also obtained. The [(125)I]Fab dimer made from normal control rabbit serum does not react with polysomes from liver at all and this preparation will not interact with polysomes extracted from tissues that do not synthesize rat serum albumin. Both anti-serine dehydratase-[(125)I]Fab monomer and dimer react with free and bound polysomes from livers of animals fed a chow diet or those fed a high 90% protein diet and given glucagon. In the latter instance, however, it is clear that the majority of the binding occurs to the bound polysomes. Furthermore, the specificity of this reaction may be further shown by the use of kidney polysomes that do not normally synthesize serine dehydratase. When these latter polysomes are isolated, even after the addition of crude and purified serine dehydratase, no reaction with anti-serine dehydratase-Fab fragments could be demonstrated. These results indicate that the reaction of the Fab fragments are specific for polysomes that synthesize rat serum albumin or rat liver serine dehydratase. Furthermore, they demonstrate that even with this high degree of specificity, some polysomes in the fraction labeled "free" are in the process of synthesizing rat serum albumin while bound polysomes to a significant, if not major, degree are the site of the synthesis of rat liver serine dehydratase.

Adaptational modification of serine and threonine metabolism in the liver to essential amino acid deficiency in rats.

It is known that plasma serine and threonine concentrations are elevated in rats chronically fed an essential amino acid deficient diet, but the underlying mechanisms including related gene expressions or serine and threonine concentrations in liver remained to be elucidated. We fed rats lysine or valine deficient diet for 4 weeks and examined the mRNA expressions of serine synthesising (3-phosphoglycerate dehydrogenase, PHGDH) and serine/threonine degrading enzymes (serine dehydratase, SDS) in the liver. Dietary deficiency induced marked elevation of hepatic serine and threonine levels associated with enhancement of PHGDH mRNA expression and repression of SDS mRNA expression. Increases in plasma serine and threonine levels due to essential amino acid deficiency in diet were caused by marked increases in hepatic serine and threonine levels. Proteolytic responses to the amino acid deficiency may be lessened by storing amino radicals as serine and inducing anorexia through elevation of threonine.

Cyclic adenosine 3',5'-monophosphate during glucose repression in the rat liver.

Intragastric administration of glucose inhibits the induction of serine dehydratase and tyrosine aminotransferase by glucagon in rat liver, but has no effect on the increase in hepatic adenosine 3',5'-monophosphate resulting from administration of glucagon. Thus, glucose repression in mammalian liver, unlike catabolite repression in microorganisms, appears to operate independently of the amounts of cyclic nucleotide in the cells.

Autogenous regulation of gene expression.

A new term, autogenous regulation, is used to describe a phenomenon that is not a new discovery but rather is newly appreciated as a mechanism common to a number of systems in both prokaryotic and eukaryotic organisms. In this mechanism the product of a structural gene regulates expression of the operon in which that structural gene resides. In many (perhaps all) cases, the regulatory gene product has several functions, since it may act not only as a regulatory protein but also as an enzyme, structural protein, or antibody, for example. In a few cases, this protein is the multimeric allosteric enzyme that catalyzes the first step of a metabolic pathway, gearing together the two most important mechanisms for controlling the biosynthesis of metabolites in bacterial cells-feedback inhibition and repression. Autogenous regulation may provide a mechanism for amplification of gene expression (84); for severe and prolonged inactivation of gene expression (85); for buffering the response of structural genes to changes in the environment (45, 52); and for maintaining a constant intracellular concentration of a protein, independent of cell size or growth rate (86). Thus, autogenous regulation provides the cell with means for accomplishing a number of different regulatory tasks, each suited to better satisfying the needs of the organism for its survival.

A regulatory element in the CHA1 promoter which confers inducibility by serine and threonine on Saccharomyces cerevisiae genes.

CHA1 of Saccharomyces cerevisiae is the gene for the catabolic L-serine (L-threonine) dehydratase, which is responsible for biodegradation of serine and threonine. We have previously shown that expression of the CHA1 gene is transcriptionally induced by serine and threonine. Northern (RNA) analysis showed that the additional presence of good nitrogen sources affects induction. This may well be due to inducer exclusion. To identify interactions of cis-acting elements with trans activators of the CHA1 promoter, we performed band shift assays of nuclear protein extracts with CHA1 promoter fragments. By this approach, we identified a protein-binding site of the CHA1 promoter. The footprint of this protein contains the ABF1-binding site consensus sequence. This in vitro binding activity is present irrespectively of CHA1 induction. By deletion analysis, two other elements of the CHA1 promoter, UAS1CHA and UAS2CHA, which are needed for induction of the CHA1 gene were identified. Each of the two sequence elements is sufficient to confer serine and threonine induction upon the CYC1 promoter when substituting its upstream activating sequence. Further, in a cha4 mutant strain which is unable to grow with serine or threonine as the sole nitrogen source, the function of UAS1CHA, as well as that of UAS2CHA, is obstructed.

Synthesis of inducible enzyme in Escherichia coli recovering from prolonged energy starvation.

A marked breakdown of ribosomes and rRNA occurs in Escherichia coli cells during prolonged deprivation of a carbon source (energy starvation). In E. coli recovering from energy starvation: (a) synthesis of RNA started immediately, total protein synthesis showed a delay of 5 to 10 minutes; (b) beta-galactosidase, tryptophanase and serine deaminase could not be induced in the first 50--70 min; (c) a lag of 60 min in the synthesis of beta-galactosidase was observed in a lac constitutive mutant of E. coli; synthesis of the constitutive enzyme malate dehydrogenase did not shown any delay. RNA synthesized in the early stages of recovery contained a higher percentage of low molecular weight molecules than RNA synthesized after 70 min of recovery or during exponential growth. Messenger RNA specific for beta-galactosidase was not synthesized for the first 50--60 min of recovery even when the specific inducer was added to the cultures.

Hormonal regulation of serine dehydratase gene expression in liver and kidney of the adrenalectomized rat.

We have previously demonstrated that glucagon but not dexamethasone could induce serine dehydratase (SDH: EC.4.2.1.13) in liver, and either glucagon or dexamethasone could induce the enzyme in kidney of normal rats. The mechanism(s) of the hormonal regulation of SDH gene expression in liver and kidney was further studied using adrenalectomized rats. Simultaneous administration of glucagon and dexamethasone induced the activity, rate of SDH synthesis, and accumulation of SDH mRNA in both liver and kidney of the rat. The increased SDH activity was reflected by changes in the amount of enzyme protein and in the rate of SDH protein synthesis, both parameters closely paralleling the changes in the levels of SDH mRNA. The rates of transcription of the SDH gene as measured in run-on experiments with isolated nuclei were also increased by the administration of these hormones. These results indicate that the expression of the SDH gene was regulated primarily at the transcriptional level under these conditions. When glucagon or dexamethasone was injected separately into adrenalectomized rats, significant increases in the levels of SDH mRNA and the rate of SDH gene transcription were observed in liver. Although glucagon was more effective than dexamethasone, both hormones were required for the maximal induction of SDH gene transcription in liver. In contrast, dexamethasone alone effectively increased the rate of SDH gene transcription in kidney.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of distinct genes with restricted expression in the somitic mesoderm in Xenopus embryo.

We have used whole-mount in situ hybridisation to identify genes expressed in the somitic mesoderm during Xenopus early development. We report here the analysis of eight genes whose expression pattern has not been described previously. They include the Xenopus homologues of eukaryotic initiation factor 2beta, methionine adenosyltransferase II, serine dehydratase, alpha-adducin, oxoglutarate dehydrogenase, fragile X mental retardation syndrome related protein 1, monocarboxylate transporter and voltage-dependent anion channel 1. Interestingly, these genes exhibit very dynamic expression pattern during early development. At early gastrula stages several genes do not show localised expression pattern, while other genes are expressed in the marginal mesoderm or in ectoderm. As development proceeds, the expression of these genes is gradually restricted to different compartments of somite. This study thus reveals an unexpected dynamic expression pattern for various genes with distinct function in vertebrates.

Identification of regions in the rat serine dehydratase gene responsible for regulation by cyclic AMP alone and in the presence of glucocorticoids.

Transcription of the rat serine dehydratase (SDH) gene is induced by glucagon, mediated by the action of cAMP. To identify the nucleotide sequences in the SDH gene responsible for this regulation, we constructed chimeric genes containing different portions of the 5' flanking region of the rat SDH gene fused to the structural sequence encoding the bacterial reporter enzyme, chloramphenicol acetyltransferase (CAT). The transcriptional activities of the fusion genes introduced into the rat hepatoma cell line 7AD-7 were assayed by measuring CAT activity in the cell lysates. Chlorophenylthio-cyclic AMP (CPT-cAMP), a potent protein kinase A activating agent, stimulated the expression of SDH-CAT fusion genes, and these inductions could be enhanced further by the addition of dexamethasone, although the glucocorticoid alone had no effect on CAT activity. Deletion analysis demonstrated that an 80 bp region located approximately 3.5 kb upstream from the transcription initiation site of the rat SDH gene was responsible for stimulation of transcription by CPT-cAMP, whereas the 120 bp region immediately upstream of the cAMP responsive element (CRE)-containing sequences is essential for the enhancement of CPT-cAMP induction by the glucocorticoid.

Hormonal control of serine dehydratase mRNA in primary cultures of adult rat hepatocytes.

The mechanism of hormonal induction of serine dehydratase [EC 4.2.1.13, SDH] was studied in primary cultures of adult rat hepatocytes by measuring the rates of syntheses of the enzyme protein and its translatable mRNA. The rate of synthesis of enzyme protein, measured as incorporation of [3H]leucine into the enzyme protein in hepatocytes, was increased 4-5 times by dexamethasone (Dex) plus glucagon. Neither hormone alone increased the rate. The increased rate induced by the two hormones was suppressed by insulin and epinephrine. The decay curves of [3H]leucine-labeled SDH showed that these hormones did not affect the rate of enzyme degradation. The level of translatable mRNA was determined by measuring cell-free synthesis of SDH in a reticulocyte lysate system. Dex plus glucagon increased the level of mRNA of SDH in hepatocytes. Insulin and epinephrine suppressed this increase without changing the rate of mRNA degradation. The level of mRNA changed in parallel with that of the rate of synthesis of the enzyme protein. These results suggest that these hormones regulate transcription of SDH, rather than its translation. After pretreatment of hepatocytes with Dex, further addition of glucagon caused more rapid induction of mRNA of SDH than addition of both hormones together. The effect of glucagon after pretreatment with Dex was inhibited by actinomycin D and alpha-amanitin, suggesting that glucagon does not affect post-transcription, but transcription per se. The requirement for both Dex and glucagon for induction of this enzyme is discussed in comparison with the requirements for either hormone alone for inductions of other gluconeogenic enzymes.

Recovery of mRNA expression of tryptophan 2,3-dioxygenase and serine dehydratase in long-term cultures of primary rat hepatocytes.

Expression of tryptophan 2,3-dioxygenase (TO) and serine dehydratase (SDH) has not previously been maintained or re-induced in long-term cultured hepatocytes. In the present study, we succeeded in inducing expression of TO and SDH mRNAs in adult rat hepatocytes cultured in serum-free L-15 medium supplemented with epidermal growth factor and 2% dimethyl sulfoxide (DMSO). After the start of culture, the expression of TO mRNA rapidly disappeared and at 96 h it was less than 10% of that at isolation. However, after the addition of 2% DMSO from 96 h, the transcript level gradually increased and reached about 40% of that of the isolated cells at day 14. In addition, the expression of TO mRNA was enhanced in cells treated with both 10(-5) M dexamethasone and 10(-7) M glucagon. In contrast, the expression of SDH mRNA decreased very rapidly and we could not detect it after 24 h of culture. Furthermore, 2% DMSO failed to induce it. However, when both 10(-5) M dexamethasone and 10(-7) M glucagon were added to the culture medium at day 9, we observed dramatic induction of SDH mRNA 24 h later. Primary hepatocytes cultured by this method could express and maintain highly differentiated hepatic functions for a long time. Thus, this in vitro system is suitable for the investigation of hepatic functions.

Interaction of adrenal and pancreatic hormones in the control of hepatic enzymes during development.

In the liver of suckling rats, the synthesis of hepatic tyrosine aminotransferase, serine dehydratase, and phosphofructokinase 2 as well as of renal beta-glucosidase is controlled by the circulating concentrations of adrenal and pancreatic hormones. Glucagon is capable of stimulating enzyme synthesis only in the presence of a steroid hormone. Dexamethasone and estradiol have been found to exert a permissive function on the inducibility of the studied enzymes by glucagon. Between the hormones of the adrenal medulla and glucagon antagonistic effects in enzyme induction were observed. Obviously, this antagonism is mediated by the alpha 1-adrenergic signal transferring system. A characteristic age dependence of enzyme induction by dexamethasone has been established. This might be correlated to alterations in the degree of methylation of the respective promoters. The methylation inhibitor 5-azacytidine influences significantly the enzyme induction by glucocorticoid hormones.

Interaction of 5-azacytidine and dexamethasone in the control of hepatic tyrosine aminotransferase.

The nucleoside analog 5-azacytidine is able to induce tyrosine aminotransferase several-fold in the liver of suckling rats. Bilateral adrenalectomy abolishes this inducing effect. The drug also decreases significantly the concentration of cytosolic glucocorticoid receptor accompanied by an increase of the glucocorticoid receptor concentration in the nuclei. The antiglucocorticoid RU 486 which abolishes the induction of tyrosine aminotransferase and serine dehydratase by dexamethasone very effectively is not able to inhibit either the induction of tyrosine aminotransferase or the translocation of the glucocorticoid receptor into the nuclei by 5-azacytidine.

Postprandial changes in portal venous free amino acids and insulin/glucagon ratios as the result of protein over-intake are not directly linked to serine dehydratase induction in rat liver irrespective of age.

The activity of hepatic serine dehydratase (SDH) increases in tandem with its gene expression when the intake of protein greatly exceeds protein requirements. The actual conditions of plasma free amino acids and pancreatic hormones in weanling and mature rats when fed SDH-inducible and non-inducible diets were examined in relation to incentive factors to secure high SDH activity from a physiological standpoint. Both weanling and mature groups differing in protein requirements were allowed free access to respective diets diverse in protein content (i.e. 25% or 50% casein diet for the former and 6% or 25% casein diet for the latter) during the dark cycle (lights-out) over a period of 1 wk. Despite the difference in protein intake among these groups, there were no conspicuous changes in the plasma concentration of the urea or total or essential amino acids. Therefore, it appears that the individual amino acids did not up regulate the gene and function expressions of SDH merely by their superabundance and subsequent disposal. Portal venous insulin concentration was far higher in mature groups than in weanling groups, although there was little difference between the two groups of the same age in terms of insulin or glucagon concentration and their ratio in abdominal vena cava blood. Accordingly, it follows that the SDH gene undergoes transcriptional regulation through a combined signaling pathway triggered by perceiving surplus protein nutrition as a whole rather than directly through already-known plasma constituents such as free amino acids or pancreatic hormones in the circulatory system.

Simultaneous induction of three catabolic enzymes in Escherichia coli.

During a simultaneous induction of three enzymes which are subject to catabolite repression (beta-galactosidase, tryptophanase and amylomaltase, or beta-galactosidase, tryptophanase and D-serine deaminase) in a batch culture, the rates of synthesis of beta-galactosidase and tryptophanase decreases, while the rates of synthesis of amylomaltase and D-serine deaminase remain unaffected. The addition of cAMP brings about a considerable increase of the rate of synthesis of D-serine deaminase and a partial synthesis rate increase of beta-galactosidase whihle the synthesis rate of tryptophanase remains lowered and the synthesis rate of amylomaltase remains unaffected. In a continuous culture beta-galactosidase, tryptophanase and D-serine deaminase are synthesized simultaneously at a maximum rate without mutual influence. The addition of cAMP increases the rate of synthesis of all three enzymes.

Catabolite repression during single and multiple induction in Escherichia coli.

Intracellular concentration of cAMP regulates the synthesis of enzymes sensitive to catabolite repression. The relationship between the single and multiple induction of beta-galactosidase (EC 3.2.1.23), L-tryptophanase (EC 4.1.99.1), D-serine deaminase (EC 4.2.1.14), L-asparaginase (EC 3.5.1.1) and L-malate dehydrogenase (EC 1.1.1.37) was studied and the effect of cAMP level on the induction in Escherichia coli Crookes (ATCC 8739) was investigated. A varying degree of catabolite repression was observed during induction of individual enzymes induced separately on different energy sources. The synthesis of l-tryptophanase was most sensitive, whereas l-asparaginase was not influenced at all. Exogenous cAMP was found to overcome partially the catabolite repression of beta-galactosidase and D-serine deaminase, both during single induction. The synthesis of l-malate dehydrogenase was negatively influenced by the multiple induction even in the presence of cAMP; on the other hand, the synthesis of l-tryptophanase was stimulated, independently of the level of the exogenous cAMP. Similarly, the activity of L-asparaginase slightly but significantly increased during the multiple induction of all five enzymes; here too the activity increase did not depend on exogenous cAMP.